Avian haemosporidians of the genera Plasmodium and Haemoproteus from resident and Neotropical migrant birds in Colombia

Avian haemosporidians of the genera Plasmodium and Haemoproteus are a group of widely distributed blood parasites that can negatively affect the fitness of their hosts. Colombia contains the greatest diversity of birds on the planet, but knowledge about the associations between haemosporidian and its avifauna is scarce and fragmented. We collected blood samples from 255 birds (203 residents and 52 neotropical migrants) belonging to 27 families and 108 species. The study was conducted in six localities in the inter-Andean valleys of the Cauca and Magdalena rivers. Parasites of the genera Plasmodium and Haemoproteus were identified in the samples by morphological and molecular analysis of a fragment of the mitochondrial gene cyt b. Among the samples, 9.3% (n = 24) were positive for Plasmodium or Haemoproteus. Co-infection with Plasmodium and Haemoproteus was found in Red-eyed Vireo﻿. Seventeen haemosporidian lineages were identified, five of which were reported for the first time in resident birds (Common Ground Dove, Checker-throated Stipplethroat, Tropical Kingbird, Pale-breasted Thrush, and Ruddy-breasted Seedeater) and one in the Summer Tanager (neotropical migrant). The research results confirm the wide diversity of haemosporidian present in tropical lowlands and the possible role of neotropical migratory birds in dissemination on haemosporidian along their migratory routes. Supplementary Information The online version contains supplementary material available at 10.1007/s00436-024-08260-8.


Introduction
Haemosporidian (Apicomplexa: Haemosporida) are a group of hemoparasites that infect a wide range of vertebrate hosts globally, including reptiles, amphibians, mammals, and birds (Ricklefs and Fallon 2002;Valkiūnas 2005;Lacorte et al. 2013;Ellis et al. 2020).Birds are susceptible to infection by mosquito-borne Plasmodium genera (Culicidae) and Haemoproteus (subgenera Parahaemoproteus and Haemoproteus), transmitted by Ceratopogonidae and Hippoboscidae flies, respectively (Valkiūnas 2005;Santiago-Alarcon et al. 2012;Toscani Field et al. 2018).Haemosporidians of the genera Plasmodium and Haemoproteus have complex life cycles with sexual reproductive stages in arthropod vectors and asexual reproduction in their avian hosts (Valkiūnas 2005).However, asexual reproduction may be disrupted in a noncompetent avian host, and the mature forms of the parasite (gametocytes), which are infectious to the vector, are not generated (i.e., abortive infection) (Valkiūnas 2005;Valkiūnas and Iezhova 2017).Infections caused by haemosporidian can have adverse effects on birds' reproductive success and survival, due to the damage caused in tissues and organs such as the spleen, liver, and brain, and consequently, affect their populations (Palinauskas et al. 2008(Palinauskas et al. , 2015;;Risely et al. 2018).
Over the past few decades, the implementation of morphological and molecular analyses has allowed the identification of a wide diversity of avian haemosporidian morphospecies and lineages in the Neotropical region (Bensch et al. 2009;González et al. 2015;Fecchio et al. 2018;2019;Ellis et al. 2020).In the neotropics, there have been reports of resident and migratory birds infected by the genera Plasmodium and Haemoproteus, which could play a significant role in the dispersal of haemosporidian throughout the Americas (Valkiūnas 2005;DeBrock et al. 2021;Lotta-Arévalo et al. 2023).In Colombia, 140 neotropical migratory bird species are recorded during the spring and fall migrations (Echeverry-Galvis et al. 2022).Infection by Plasmodium (Haemamoeba) cathemerium, Haemoproteus (Parahaemoproteus) vireonis, or Haemoproteus (Parahaemoproteus) coatneyi has been detected in birds of the Vireonidae, Turdidae, or Cardinalidae families (González et al. 2015;Pulgarín-R et al. 2019).In Colombia, there is a limited and fragmented knowledge about haemosporidians and their interaction with wild birds, with studies mainly conducted in localities of the Caribbean, Orinoco, and High Andean regions (González et al. 2015;Alvarez-Londoño et al. 2022;Lotta-Arévalo et al. 2023).This lack of information hinders the understanding of birds' role in the epidemiology of Plasmodium and Haemoproteus.
In this context, and considering that the regions encompassing the inter-Andean valleys of the Cauca and Magdalena rivers are include in the migration routes of at least 83 species of neotropical migratory birds (Fierro-Calderón and Eusse 2010; Naranjo et al. 2012), this region could serve as a "transmission zone" where resident-migratory bird species and haemosporidians of South and North American origin converge (Moens and Pérez-Tris 2016;Fecchio et al. 2018).This research aimed to determine the diversity and prevalence of Plasmodium and Haemoproteus in neotropical resident and migratory wild birds at six locations in the inter-Andean valleys of the Cauca and Magdalena rivers.

Study area
The study was conducted in six localities within the department of Caldas, Colombia (Latitude: 4.07194, Longitude: − 75.95722).The localities are situated in the inter-Andean valleys of the Cauca (Municipality of Palestina) and Magdalena rivers (Municipalities of La Dorada, Samaná and Victoria) (Fig. 1).The six localities are found within an elevation range of 126 and 1050 m a.s.l.They experience a bimodal rainfall pattern (average annual precipitation ranging from 2000 to 4000 mm).The highest rainfall occurs between March and June and between September and December.The average temperature ranges between 20 and 27 °C (Martínez-Sánchez et al. 2018).The sampled localities in the inter-Andean valley of the Magdalena River are farms dedicated to the production of avocado (Persea americana Mill.) or cocoa (Theobroma cacao L.), or buffalo rearing (Bubalus bubalis L.) and two secondary forests.The two locations that were sampled in the inter-Andean valley of the Cauca River correspond to two farms that produce cocoa (T.cacao) or citrus (Citrus spp.) production (Table 1).

Collection and processing of bird blood samples
Wild birds were captured from September 2021 to February 2022 using six mist nets (12 × 2.5 m × 36 mm) at each location for five consecutive days, with a total sampling intensity of 1980 h/net.The nets were operated between 06:00 and 17:00 h.Captured birds were marked with a small cut on the first rectrix to avoid recounts and were subsequently released at the location where they were captured (Martínez-Sánchez et al. 2018).The bird taxonomy followed the nomenclature of Remsen et al. (2023).Residency status (neotropical migratory or resident) was determined according to Echeverry-Galvis et al. (2022).A blood sample (approximately 20 to 50 μl) was obtained from each captured bird through brachial vein puncture using 25G and 27G gauge needles (Busi et al. 2020).To obtain the hummingbird blood sample, a small cut was made near the fingernail (Owen 2011).The samples were used to create blood smears (at least two per bird), fixed in absolute methanol for 5 min, and stained with 5% GIEMSA solution, pH 7.2 for 45 min (Alvarez-Londoño et al. 2022).The remaining blood was also deposited on FTA cards (Flinders Technology Associates) for subsequent molecular analysis.

Morphological analysis
For morphological identification of haemosporidians, blood smears were analyzed using a light microscope, specifically an Olympus BX43 optical microscope, with 40 × and 100 × objectives; approximately 100-150 fields were visualized per objective (Valkiūnas 2005).The parasites were identified using specialized taxonomic guides (Valkiūnas 2005;Valkiūnas and Iezhova 2018;2022).Images of the parasites were captured using an Olympus DP28 digital camera and edited with Olympus CellSens Standard v3.22.11software.

Molecular analysis
DNA extraction from blood samples stored on FTA cards was performed using the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, USA) following the manufacturer's instructions.To detect Plasmodium and Haemoproteus DNA, nested Polymerase Chain Reaction (PCR) testing was conducted on a fragment of a cytochrome   (Pacheco et al. 2018).One microliter of the initial PCR was utilized for the second PCR, utilizing the HaemF/HaemR2 primers that amplify a 480 bp fragment for Plasmodium and Haemoproteus (Hellgren et al. 2004).
To determine Plasmodium DNA, a nested PCR was conducted using the initial PCR amplicon and primers AE983/ AE985 which amplify a 580 bp fragment (Pacheco et al. 2018).Similarly, a nested PCR was performed using the initial PCR amplicon and primers AE980/AE982 to determine Haemoproteus DNA, resulting in amplification of a 346 bp fragment (Pacheco et al. 2018) Sequences that had at least one nucleotide difference with the MalAvi sequences were considered new lineages and were named according to the nomenclature proposed by Bensch et al. (2009).Phylogenetic reconstruction was conducted using Bayesian inference on the identified lineages of Plasmodium and Haemoproteus.The alignments included our sequences and sequences of the cyt b gene obtained from the MalAvi database which were linked to morphospecies, and a sequence of Leucocytozoon buteonis BUTREG01 [DQ177264] was used as an outgroup.The total length of the alignments was 480 bp.The General Time Reversible model with invariant sites and gamma distribution (GTR + I + G) was selected according to the corrected Akaike information criterion using jModeltest v.2.1.6(Darriba et al. 2012).Bayesian analysis was carried out using MrBayes v3.2.7a (Ronquist and Huelsenbeck 2003) via the CIPRESS Science Gateway v3.3 (Miller et al. 2010).Two independent Markov and Monte Carlo chains (MCMC) were used simultaneously for a total of 15 million generations, with four chains sampled every 1000 generations.Convergence was evaluated by calculating the mean and standard deviation of the frequencies divided between the two runs below 0.01 and graphically using Tracer (Rambaut and Drummond 2007).A total of 25% of the trees were discarded as run periods, and the remaining trees were used to create a consensus tree applying a 50% majority rule.The phylogeny was visualized using FigTree v1.3.1 (Rambaut 2013).All obtained sequences were deposited in MalAvi and GenBank.The percent prevalence of Plasmodium or Haemoproteus in birds was determined using the following equation [(number of infected individuals / number of individuals examined) × 100] ( Bush et al. 1997).Molecularly positive samples that did not present forms of the parasite in the blood were not included in the prevalence estimation (Alvarez-Londoño et al. 2022).

Discussion
The prevalence of Plasmodium spp. or Haemoproteus spp. was similar to that established in other localities in Colombia (Rodríguez and Matta 2001;Valkiūnas et al. 2003;Basto et al. 2006;Alvarez-Londoño et al. 2022).It has been suggested that the prevalence of these haemosporidians in birds seems to be determined by habitat characteristics (e.g., lentic water bodies, ponds, or forest type) that support the development, abundance, and diversity of their vectors or by  (González et al. 2015;Alvarez-Londoño et al. 2022).Particularly, one of the locations with the highest number of Plasmodium spp.infected birds was the Granja Luker.On this farm, pools are formed as a result of the crop drainage network, and there are bromeliads attached to trees that serve as habitat for mosquitoes of the genus Wyeomyia, recognized vectors of avian Plasmodium (Bensch et al. 2009;Morcillo et al. 2023).
Seventy percent of the Plasmodium lineages reported in our study have previously been detected in America, Europe, Asia, or Australia (Bensch et al. 2009;Fig. 4).However, some of these Plasmodium lineages were documented for the first time in Colombia within resident or neotropical migratory birds.In relation to this, we consider it as an extension of the geographic distribution range across the Americas for the SETAUD23 and TACTHA01 lineages of Plasmodium sp.detected in House Wren and Scarlet Tanager, respectively.Both lineages have been previously reported in North America in neotropical migratory birds wintering in Colombia (Bensch et al. 2009;Fig. 4).We recorded the geographic expansion in the South American distribution of the VIOLI03 and EMBHER01 lineages of Plasmodium sp., as well as the EUPMAC01 lineages of H. (P.) witti, ATLBRU01 of Haemoproteus sp., TAN-CYA01 of H. (P.) erythrogravidus.These lineages had previously been recorded only in other countries of the Americas, such as the USA, Guyana, Peru, or Brazil, in both passerine and non-passerine birds (Beadell et al. 2006;Durrant et al. 2006;Bensch et al. 2009;Fecchio et al. 2019).Likewise, we report the presence of P. (H.) matutinum LINN1 in Colombia infected to a migratory Graycheeked Thrush.This Plasmodium morphospecies has previously been recorded in North America, Europe, Asia, and Australia, infecting birds of various orders including Gruiformes, Charadriiformes, Strigiformes, or Passeriformes (Bensch et al. 2009;Fig. 4).In particular, P. (H.) matutinum has been observed in birds on the east coast of the USA (states of Michigan and New York) (Bensch et al. 2009).These localities are part of the migration routes of the Gray-cheeked Thrush from their breeding grounds in Asia (northeastern Siberia) and North America (from Alaska to the East Coast) (Udvardy 1994).This suggests that neotropical migratory birds may play a key role in the dispersal of parasites of the genus Plasmodium between temperate and tropical regions of the Americas (Hellgren et al. 2007;Fecchio et al. 2018).We reported six new lineages of haemosporidians infecting resident and migratory birds, of which three lineages of Haemoproteus (COL-PAS11, TYRMEL03, and SPOMIN01) and one lineage of Plasmodium (TULEU09) were related to a morphospecies through morphological analysis.The TULEU09 lineage of P. (N.) unalis recorded in the Pale-breasted Thrush appears to be related to the TFUS06 lineage of P. (N.) unalis that has been previously documented in the Great Thrush (Turdus fuscater) in Colombia, as well as four other species of the genus Turdus in the Atlantic forest of Brazil (Turdus rufiventris, T. leucomelas, Turdus albicollis, and Turdus flavipes) (Mantilla et al. 2013;Tostes et al. 2018;Fig. 4).In this regard, it has been suggested that there is a high degree of intraspecific polymorphism, and a wide diversity of host species are present within this parasite species (Mantilla et al. 2013;Vanstreels et al. 2015;Tostes et al. 2018).The newly discovered EPIFUL01 lineage of Plasmodium sp.detected in the Checker-throated Stipplethroat is part of a polytomy of diverse Plasmodium morphospecies (Fig. 4).Therefore, this new lineage could be another Plasmodium species, but further studies on this host are required to confirm.The new Plasmodium sp.lineage PIRUB04 found in the Summer Tanager was related to the lineages EMBHER01 in the Sooty-ant Tanager, SETAUD23 in the House Wren, TACTHA01 in the Scarlet Tanager, and PADOM09 in the Bananaquit (evolutionary distances of < 1.7%; Table S1).The latter lineages were related to the Plasmodium (H.) cathemerium morphospecies (ZONCAP15), which was previously documented to infect the Rufous-collared Sparrow (Zonotrichia capensis) in Ecuador (Fig. 4).Moreover, the new COLPAS11 lineage of H. (H.) paramultipigmentatus was detected in Common-ground Dove in the Magdalena River Valley.This lineage grouped with the COLPAS03 and COL-BUC01 lineages belonging to the morphospecies H. (H.) paramultipigmentatus and Haemoproteus (Haemoproteus) multipigmentatus, respectively (Fig. 4).These lineages are closely associated with birds from the Columbidae family, with species distributed in Mexico, Ecuador, Brazil, Venezuela, and recently reported in Colombia (Valkiūnas et al. 2010;Lotta-Arévalo et al. 2023).Another new lineage, TYRMEL03 of H. (P.) tyranni, was detected in a Tropical Kingbird in the Cauca River Valley.This lineage was associated with the MYMAC03 lineage reported in this study in another individual from the Tropical Kingbird (Fig. 4).The MYMAC03 lineage had previously been detected only through molecular methods in another member of the Tyrannidae family (Myiodynastes maculatus) in Brazil (Ferreira et al. 2017).The morphospecies H. (P.) tyranni does not have an associated lineage in the MalAvi database; however, it has a significantly higher number of records in North America (the USA and Canada), while in the rest of the Americas, it has only been previously recorded in Panama, Brazil, and Venezuela, infecting mainly birds of the family Tyrannidae (e.g., Pitangus sulphuratus and T. melancholicus) (Valkiūnas 2005;Valera et al. 2019;da Silva et al. 2022).Therefore, we consider that the record of H. (P.) tyranni increases its distribution range in South America and provides a lineage associated with this morphospecies.The new lineage SPOMIN01 of H. (P.) nucleocentralis was closely related to the lineage TANDES01 of the same morphospecies (detected only in Brazil).These two lineages have only been reported in two species of the Thraupidae family (Sporophila minuta and Tangara desmaresti) (Anjos et al. 2021).This study contributes to the knowledge of the diversity of Plasmodium and Haemoproteus lineages and species present in neotropical resident and migratory birds in tropical lowlands.

Fig. 4
Fig. 4 Bayesian phylogeny of Haemoproteus spp.and Plasmodium spp.based on 117 lineages of cytochrome b gene.Morphological species names followed by MalAvi lineage, GenBank accession number within square brackets, and the country where the sequences are reported.The colored rectangles correspond to the geographic regions where the lineages from the MalAvi database have been

Table 1
Descriptions of the localities in the department of Caldas where birds were sampled, Colombia.The localities correspond to the numbers presented in Fig.1 . Samples positive by PCR with primers AE983/AE985 and primers AE980/AE982 were considered coinfections.All PCR reactions were conducted with both a negative control (H 2 Odd) and positive control (Haemoproteus columbae) (Alvarez-Londoño et al. 2022).To visualize PCR products, horizontal electrophoresis was carried out using 1% agarose gels with 1X TBE buffer, stained with SYBR SAFE (Thermo Fisher Scientific, Waltham, USA), and visualized in UV photodocumenter.Positive samples for HaemF/HaemR2 were sent to Macrogen (Seoul, South Korea) for purification and Sanger sequencing.The obtained sequences were edited in the Geneious Prime program (2023.0.4.https:// www.genei ous.com/) and aligned in the MEGA11 software (Tamura et al. 2021).To identify Plasmodium and Haemoproteus genetic lineages, we compared our sequences with sequences deposited in the public databases MalAvi (http:// 130. 235.244.92/ Malavi/, Bensch et al. 2009) and GenBank (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).

Table 2
Avian hosts positive with Plasmodium spp.andHaemoproteus spp. in the department of Caldas.The GenBank accession codes in bold correspond to the sequences obtained in this study.Localities where birds were sampled in the department of Caldas, Colombia.The localities correspond to the numbers presented in Fig.1*New haemosporidian lineages reported in the study ˆNeotropical migrant bird S Presence of blood forms, PCR negative a Molecularly positive samples that did not present forms of the parasite in the blood b Unidentified lineage (low quality sequence)

Table 3
Non-infected bird species by haemosporidian parasites caught in the department of Caldas-Colombia (the localities correspond to the numbers presented in Fig.1)